Eukaryotic lipid metabolic pathway is essential for functional chloroplasts and CO2 and light responses in Arabidopsis guard cells Juntaro Negia,1, Shintaro Munemasab,2, Boseok Songa, Ryosuke Tadakumaa, Mayumi Fujitaa, Tamar Azoulay-Shemerb, Cawas B. Engineerb, Kensuke Kusumia, Ikuo Nishidac, Julian I. Schroederb, and Koh Ibaa,1 aDepartment of Biology, Faculty of Science, Kyushu University, 819-0395 Fukuoka, Japan; bCell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093; and cGraduate School of Science and Engineering, Saitama University, 338-8570 Saitama, Japan Edited by Krishna K. Niyogi, Howard Hughes Medical Institute and University of California, Berkeley, CA, and approved July 27, 2018 (received for review June 22, 2018) Stomatal guard cells develop unique chloroplasts in land plant serve as a substrate for glycolipid synthesis (10–12) (SI Appendix, species. However, the developmental mechanisms and function of Fig. S1). The prokaryotic pathway contributes to chloroplast PG chloroplasts in guard cells remain unclear. In seed plants, chloroplast synthesis in all plants, and to glycolipid synthesis only in the so-called membrane lipids are synthesized via two pathways: the prokaryotic 16:3 plants, which contain 16:3 (acyl carbons: double bonds) in the and eukaryotic pathways. Here we report the central contribution sn-2 position of MGDG. In contrast, the eukaryotic pathway con- of endoplasmic reticulum (ER)-derived chloroplast lipids, which are tributes to the glycolipid synthesis in all plants. MGDG synthesized synthesized through the eukaryotic lipid metabolic pathway, in the via the eukaryotic pathway contains C18 fatty acids, most abundantly development of functional guard cell chloroplasts. We gained 18:3, in the sn-2 position, and those plants, including pea (Pisum insight into this pathway by isolating and examining an Arabidopsis sativum)andrice(Oryza sativa), that exclusively use the eukaryotic mutant, gles1 (green less stomata 1), which had achlorophyllous pathway in plastid glycolipid synthesis have been referred to as 18:3 stomatal guard cells and impaired stomatal responses to CO and 2 plants. In 16:3 plants, the contribution of the prokaryotic and the light. The GLES1 gene encodes a small glycine-rich protein, which is eukaryotic pathways varies among different plant species (13). The a putative regulatory component of the trigalactosyldiacylglycerol (TGD) protein complex that mediates ER-to-chloroplast lipid trans- contribution of the lipid flux through the prokaryotic pathway is port via the eukaryotic pathway. Lipidomic analysis revealed that in the highest in nonseed plants, such as mosses and ferns, whereas it the wild type, the prokaryotic pathway is dysfunctional, specifically has been estimated as 38% in Arabidopsis (14). Moreover, even in in guard cells, whereas in gles1 guard cells, the eukaryotic pathway the same16:3 plant species, the prokaryotic and the eukaryotic is also abrogated. CO2-induced stomatal closing and activation of pathways do not necessarily work at a fixed proportion in all tissues. guard cell S-type anion channels that drive stomatal closure were For example, in Arabidopsis, the prokaryotic pathway appears to be disrupted in gles1 guard cells. In conclusion, the eukaryotic lipid strongly diminished during embryo development (15–18). Although pathway plays an essential role in the development of a sensing/ each of the prokaryotic and the eukaryotic pathways produces lipid signaling machinery for CO2 and light in guard cell chloroplasts. Significance stomata | chloroplast | lipid metabolism | CO2 | Arabidopsis Guard cells have photosynthetically active chloroplasts in most tomatal pores allow an influx of CO2 in exchange for tran- plant species. However, the significance of their existence in Sspirational water loss. The stomatal aperture is regulated by guard cells or their developmental mechanisms is unknown. environmental and physiological factors, especially CO2, the Here, through a forward-genetic approach, we have identified plant hormone abscisic acid (ABA), humidity, light, and ozone a key feature and a function of guard cell chloroplasts. We (1–4). Chloroplasts in the guard cells of stomata have been observed that a mutation that impaired chloroplast biogenesis proposed to play an important role in osmoregulatory mecha- in guard cells also disrupted the regulation of stomatal move- nisms mediating stomatal movements (5, 6), although their ments by CO2 and light. We demonstrated that guard cell functions have been a subject of debate. To date, studies on chloroplasts, compared with those in mesophyll cells, display a guard cell chloroplasts have largely focused on their photosyn- unique lipid metabolism, in which the prokaryotic pathway is thetic activities (7–9), whereas the relevance of lipid synthesis diminished and the eukaryotic pathway gains control. Our remains poorly investigated. findings highlight the importance of the eukaryotic pathway Chloroplast development accompanies the biogenesis of thyla- for developing functional chloroplasts in guard cells. koid membranes, which requires the coordinated synthesis of membrane proteins and glycerolipids. The thylakoid membranes Author contributions: J.N. and K.I. designed research; J.N., S.M., B.S., R.T., M.F., T.A.-S., C.B.E., and K.K. performed research; J.N. contributed new reagents/analytic tools; J.N., consist of the glycolipids monogalactosyldiacylglycerol (MGDG), I.N., J.I.S., and K.I. analyzed data; and J.N., I.N., J.I.S., and K.I. wrote the paper. digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylgly- The authors declare no conflict of interest. cerol and the phospholipid phosphatidylglycerol (PG). Fatty acids This article is a PNAS Direct Submission. are exclusively synthesized de novo within plastids, but the assembly This open access article is distributed under Creative Commons Attribution-NonCommercial- of fatty acids into the glycerolipids of thylakoid membranes occurs NoDerivatives License 4.0 (CC BY-NC-ND). via two distinct pathways: the prokaryotic pathway and the eukary- 1 – To whom correspondence may be addressed. Email: [email protected] otic pathway (10 12). In the prokaryotic pathway, all reaction steps or [email protected]. take place within the chloroplast (hence called the plastidial path- 2Present address: Graduate School of Environmental and Life Science, Faculty of Agricul- way), whereas in the eukaryotic pathway or the cooperative pathway, ture, Okayama University, 700-8530 Okayama, Japan. fatty acids are exported from the chloroplast to the cytosol to be This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. assembled into glycerolipids in the endoplasmic reticulum (ER). 1073/pnas.1810458115/-/DCSupplemental. Some of the ER-localized glycerolipids return to the chloroplast to Published online August 20, 2018. 9038–9043 | PNAS | September 4, 2018 | vol. 115 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1810458115 Downloaded by guest on September 30, 2021 molecular species with distinct fatty acid distribution, the physio- it has been reported that triacylglycerols stored in guard cells are logical significance of such metabolic divergence remains unclear. used to produce ATP required for light-induced stomatal opening The eukaryotic pathway requires lipid transfer from the ER to (25). However, the distinct roles of prokaryotic and eukaryotic lipid chloroplasts. The trigalactosyldiacylglycerol (TGD) protein com- metabolic pathways in guard cells have not been understood. plex has been identified as playing a major role in the lipid transfer In this study, we have found, through a forward-genetic approach, from the ER to chloroplasts (19). The TGD protein complex itself that lipid synthesis in guard cells is distinct from that in mesophyll is an ABC-type transporter consisting of three subunits: TGD1, cells, and that the prokaryotic pathway is extensively retarded in TGD2, and TGD3. TGD1 is an ABC transporter permease lo- Arabidopsis guard cells. As a consequence, lipid transfer from ER to cated in the inner envelope membranes of chloroplast, TGD3 is chloroplast through the eukaryotic pathway gains more significance an ATPase or a nucleotide-binding protein facing to stroma, and and seems essential for guard cell chloroplast development and for TGD2 is a substrate-binding protein, which is suggested to bind a stomatal CO2 and light responses in Arabidopsis guard cells. lipid substrate in outer envelope membranes to deliver it to TGD1. An additional subunit, TGD4, is located in outer envelope Results and Discussion membranes to play a tethering role between the ER and the Isolation of Arabidopsis gles1 Mutant That Develops Abnormal chloroplast envelope (20, 21). Furthermore, TGD5, a small Chloroplasts in Guard Cells. Previously, we isolated a CO2-insensitive glycine-rich protein, has been hypothesized to play a regulatory mutant line (cdi6) from an M2 population of ∼30,000 ethyl role in the ER-to-chloroplast lipid transfer (22). methanesulfonate-mutagenized Arabidopsis plants, using leaf in- To date, the studies of lipid metabolism in guard cells have been frared imaging thermography (3). This technology enabled us to confined to an 18:3 plant species, Vicia faba (23, 24). Using [14C] isolate a number of mutants that showed abnormal leaf temperature acetate labeling, guard cell protoplasts from V. faba have been resulting from malfunction in stomatal